SUMMARY Eukaryotes have evolved a number of strategies to manage misfolded and aggregated proteins and therein maintain protein homeostasis (proteostasis) in response to cellular stress. These include proteolytic pathways such as the ubiquitin proteasome system and restorative pathways in which damaged proteins are rescued by molecular chaperones and disaggregases. In humans, a breakdown in proteostasis is linked with diverse pathologies including neurodegenerative diseases and cancer. A cellular hallmark of these diseases is the presence of cytoplasmic and/or nuclear inclusion bodies which for many years were thought to contribute to disease establishment and progression. However, a growing body of evidence reveals that inclusion bodies form as part of a highly-orchestrated cellular protective pathway called spatial protein quality control (SPQC), in which damaged proteins are sequestered and confined to discrete quality control compartments to be processed later by the cell. The long-term goals of my research program are to determine in mechanistic detail how, where, when, and why damaged proteins are recruited to inclusion bodies and to understand the cellular consequences of dysfunction in this pathway. We are using the filamentous fungus Aspergillus nidulans as an innovative model system to understand SPQC in highly polarized cells, and we have previously demonstrated the importance of the microtubule-based transport system in organizing aggregated proteins following heat shock. The overall Aims in this application are to 1) determine the ultimate cellular fates of endogenous misfolded proteins following their spatial confinement to inclusion bodies and 2) further elucidate the molecular mechanisms of SPQC in highly polarized A. nidulans cells. In Aim 1, we will first identify the endogenous substrates of cytoplasmic and nuclear inclusion bodies in our system using innovative proteomics approaches. Then, using photoconvertible fluorescent proteins, we will track the spatiotemporal fates of these endogenous substrates under normal and perturbed conditions to uncover the extent of functional interplay between major proteostasis pathways and SPQC. In Aim 2, we will test the hypothesis that protein aggregates hitchhike on moving early endosomes for transportation to protein quality control compartments and examine the contribution of microtubule dynamics to aggregate reorganization and SPQC. In addition, we will use super- resolution fluorescence microscopy to resolve the nanoscale organization of cytoplasmic inclusion bodies in our system, therein gaining new insight into their form and function. Lastly, we will employ an unbiased genome-wide fluorescence microscopy-based screen to identify novel regulators of SPQC in highly polarized Aspergillus cells. Consistent with the goals of the AREA program, this research will provide a wealth of opportunities for hands-on undergraduate research involvement and exposure, while pushing our mechanistic understanding of this emerging proteostasis pathway forward.